EP0699904A1 - Method for detecting organopathyinducing factors - Google Patents

Method for detecting organopathyinducing factors Download PDF

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Publication number
EP0699904A1
EP0699904A1 EP95113759A EP95113759A EP0699904A1 EP 0699904 A1 EP0699904 A1 EP 0699904A1 EP 95113759 A EP95113759 A EP 95113759A EP 95113759 A EP95113759 A EP 95113759A EP 0699904 A1 EP0699904 A1 EP 0699904A1
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organopathy
serum
cells
cultured
organ
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English (en)
French (fr)
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Toshimasa Yoshioka
Katsumi Ito
Masaru c/o Kureha Chem. Ind. Co. Ltd. Motojima
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Kureha Corp
Tokyo Womens Medical University
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Kureha Corp
Tokyo Womens Medical University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

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  • the present invention relates to a method for detecting organopathy-inducing factors, which can be used for the diagnosis of organopathy. More particularly, it relates to a method for obtaining indicators of organopathy by Electron Paramagnetic Resonance (EPR) (or Electron Spin Resonace, ESR) or the like, which enables one to detect organopathy-inducing factors and to diagnose even latent organopathy at an early stage.
  • EPR Electron Paramagnetic Resonance
  • ESR Electron Spin Resonace
  • organopathy Hitherto, clinical diagnosis of organopathy was not carried out until the organopathy actually became clear.
  • organ incompetency the parameters indicating organ functions are abnormal in value after the organopathy has advanced to a certain extent.
  • organopathy is manifested, it is often impossible to stop the advance of the disorder.
  • clinical diagnosis of renal failure can be done by a rise in serum creatinine, which is an index of glomerular filtration rate (i.e. renal function), or by histological changes in the kidney.
  • serum creatinine which is an index of glomerular filtration rate (i.e. renal function)
  • histological changes in the kidney In particular, in acute renal failure, it is often impossible to stop the advance of disorder when renal dysfunction has become clinical (such oliguria, edema).
  • ROS reactive oxygen species
  • H2O2 hydrogen peroxide
  • HO ⁇ hydroxyl radicals
  • HOO ⁇ hydroperoxyl radicals
  • ROS has a close relationship with various organopathies such as aging, cancer, inflammation, ischemic organopathy, arteriosclerosis, and drug-induced diseases [Kunie Nakamura, et al., Kassei-Sanso-Furii-Rajikaru (J.Act. Oxyg. Free Rad.), 3(1); 63-70, 1992]. Research on the relationship between ROS and disease, however, related to the mechanism of the disease. Application of said relationship has not been known.
  • the present inventors engaged in intensive research to develop a screening method for detecting organopathy in its early stage.
  • Blood contains causative substances (for example, endotoxins, nephrotoxic drugs, immunocomplexes, etc.) inducing, for example, nephropathy, that is, nephropathy-inducing factors.
  • causative substances for example, endotoxins, nephrotoxic drugs, immunocomplexes, etc.
  • nephropathy that is, nephropathy-inducing factors.
  • the inventors discovered that it is possible to detect nephropathy-inducing factors contained in the blood, using the fact that ROS is released from kidney cells at the early stage of nephropathy, namely, by applying serum or plasma to cultured kidney cells to thereby cause the serum or plasma to act on the cells, and measuring the release of ROS from the cells by means of EPR or the like.
  • organopathy-inducing factors can be detected by causing serum or plasma to act on specific cultured organ cells other than cultured kidney cells and measuring the release of ROS from the cells by means of EPR or the like.
  • the present invention is based on these discoveries.
  • the object of the present invention is to provide a method for detecting organopathy in its early stage.
  • a novel method for detecting organopathy-inducing factors in a serum or plasma sample comprising the following steps (1) to (3):
  • organopathy-inducing factor means a causative substance which is present in the blood and induces organopathy.
  • the organ is not particularly limited and may be the kidney, liver, heart, lung, digestive tract (stomach and intestines), nervous system (such as brain), or the like.
  • the kind of the disorder is also not particularly limited. Accordingly, for example, a nephropathy-inducing factor is a causative substance which is present in the blood and induces nephropathy, for example, an endotoxin, nephrotoxic drug, immunocomplex, or the like.
  • organopathy-inducing factors there may be mentioned hepatopathy-inducing factors, cardiopathy-inducing factors, pulmopathy-inducing factors, gastroenteropathy-inducing factors, encephalopathy-inducing factors, or the like.
  • the method of the present invention may be applied to any causative substance which is present in the blood and induces organopathy. Accordingly, the organopathy-inducing factors in the present invention naturally include substances which are not yet known to exist at the present time.
  • Step (1) Cultivation of Cultured Organ Cell Lines In Presence of Serum or Plasma to be Examined
  • Step (1) comprises adding a sample, that is, a serum or plasma, to the cultured cell lines and further cultivating the cell lines whereby reactive oxygen species (ROS) are released in the culture medium.
  • a sample that is, a serum or plasma
  • the sample either of serum or plasma may be used.
  • the serum or plasma added as the sample is not particularly limited, so long as it possibly might contain an organopathy-inducing factor to be measured.
  • the sample may be obtained, for example, by separating the serum or plasma from blood taken from humans or animals by ordinary methods.
  • the amount of serum or plasma added may be 0.1 to 1.0 ml, preferably 0.4 to 0.6 ml, per one examination with respect to the cell numbers (1 x 106 to 10 x 106) used in a usual examination. If the amount of serum or plasma added is less than 0.1 ml, the reaction becomes weak and the sensitivity of detection falls. If the amount of serum or plasma added exceeds 1.0 ml, it becomes necessary to increase the amount of the trapping agent used, but the sensitivity does not rise. Thus, it is of no practical use.
  • cultured organ cell lines cultured cell lines derived from the organ same as the organ in which the disorder is caused by the organopathy-inducing factor to be examined, that is, cultured cell lines derived from the organ corresponding to the organ for which the disorder is to be determined.
  • a cell strain obtained by taking cells from the organ tissue in an animal and subculturing them in accordance with the ordinary tissue culture method may be used. It is preferable to use a so-called immortalized cell strain which permanently sustains considerable growth activity to the extent that the first generation cells possess and further does not lose the form and function inherent to the cells even after the subculturing. Such cells for some organs are also commercially available.
  • the immortalized cell strain as follows: (1) the method comprising introducing into animal cells oncogene, such as ras or c-myc, or DNA type tumor virus gene, such as adenovirus E1A, SV40 virus, human papilloma virus (HPV16), or their tumor antigen (T-antigen) gene, and subculturing the transformant; (2) the method comprising preparing a transgenic animal having a oncotic gene or its T-antigen gene stably incorporated in a part of the chromosome, obtaining the primary cells from the transgenic animal which holds a part of the oncotic gene in the somatic cells and/or germ cells already at the time of ontogeny, and subculturing the same; (3) the method comprising introducing the temperature-sensitive mutant SV40 large T-antigen gene into a totipotent cell of a mammal, obtaining the transgenic animals by normal reproduction of said mammal, taking the cells from
  • kidney cells As the cultured kidney cells it is preferable to use cell lines from endothelial cells, immortalized bovine glomerular endothelial cells, glomerular mesangium cells, LLCPK1 cells (a cell line derived from renal tubules, i.e., the common loci of acute renal failure), or the like. It is particularly preferable to use the immortalized bovine glomerular endothelial cell lines, because the cultivation thereof is simple and the cell lines can be subcultured substantially permanently.
  • endothelial cells immortalized bovine glomerular endothelial cells, glomerular mesangium cells, LLCPK1 cells (a cell line derived from renal tubules, i.e., the common loci of acute renal failure), or the like.
  • LLCPK1 cells a cell line derived from renal tubules, i.e., the common loci of acute renal failure
  • the cell cultivation in the step (1) of the present invention may be performed by ordinary methods in an ordinary cell culture facility, for example, a clean bench (SCV1303EC2A; Hitachi, Tokyo), or the like.
  • the subculturing cells may be maintained in a culture flask (for example, 75 cm; plastic flask made by Corning or Falcon).
  • the culture medium and culture conditions may be suitably selected in accordance with the organ tissue used. For example, when cultivating renal glomerular cells, it is preferable to use RPMI1640, or the like as the culture medium.
  • the cultured cell lines used may be subcultured on a culture plate (for example, having six wells of a diameter of 35 mm, e.g. 6-well plate; made by Corning or Falcon) coated with, preferably, gelatin (for example, made by Sigma)
  • a culture plate for example, having six wells of a diameter of 35 mm, e.g. 6-well plate; made by Corning or Falcon
  • gelatin for example, made by Sigma
  • the cells for subculture in the culture flask are separated with an enzyme (for example, trypsin), suspended in the culture medium, then divided on the culture plate. After, 24 to 72 hours, the cell density becomes constant (approximately 100,000 to 500,000 cells/well on the 6-well plate), the cells are further cultivated for 12 to 72 hours in a maintaining medium (for example, RPMI 1640 containing 0.5% fetal bovine serum).
  • a maintaining medium for example, RPMI 1640 containing 0.5% fetal bovine serum
  • Cells from the cultured cell lines can be stimulated with a serum or plasma sample by the following method: For example, the cells on the culture plate at the step (1) of the present invention first are washed with a medium such as Dulbecco's PBS (D-PBS including calcium and magnesium; Gibco) or the like. Then, a given amount of D-PBS and sample are added to each plate. Typically, 0.4 ml of D-PBS and 0.2 ml of sample are applied to a well of 6-well plate. Subsequently, the cells are cultivated in a CO2 incubator (a normal incubator for cell culture, for example, WJ-3C, Hirasawa Seisakusho, Tokyo) or the like. The preferable cultivation or incubating period is 0.5 to 5 hours, more preferably 1 to 2 hours, most preferably 1 hour. If the incubation period is shorter than 0.5 hour, no reaction occurs, while if it is longer than 5 hours, the cells sometimes die.
  • a medium such as Dulbecco'
  • ROS includes a superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen, and so on produced by the incomplete reduction of the oxygen molecules.
  • Step (2) Addition of Spin-Trapping Agent
  • Step (2) comprises adding a spin-trapping agent to the culture medium obtained by the above step (1).
  • the unstable free radicals that is, ROS
  • the spin-trapping agent is reacted with the spin-trapping agent to convert them to stable, easily measurable spin adducts.
  • the spin-trapping agent is not particularly limited so long as it can convert ROS to spin adducts.
  • DMPO 5,5-dimethyl-1-pyrroline-N-oxide
  • MNP 2-methyl-2-nitrosopropane dimer
  • PBSN ⁇ -phenyl-N-t-butylnitron
  • the spin-trapping agent is preferably added in excess so that all the ROS in the culture medium can be converted to spin adducts.
  • the reaction may be carried out 1 to 60 minutes, preferably 10 to 30 minutes, after adding the spin-trapping agent. If the reaction time is less than 1 minute, conversion to spin adducts is insufficient, while if the reaction time is over 60 minutes, the spin-trapping agent may cause cell dysfunction.
  • Step (3) comprises measuring the concentration of radicals in the culture medium obtained at the step (2), for example, using an EPR apparatus.
  • the frequency may be any of 1.2 GHz (L-band), 9.4 GHz (X-band), 24 GHz (K-band), 36 GHz (Q-band), or the like, but the X-band is preferable.
  • the spin measurement standard for example, 4-hydroxyl-2,2,6,6-tetramethyl-1-piperidine-N-oxyl (4-hydroxy-TEMPO) may be used.
  • the height obtained by integrating the resulting spectrum twice is the concentration of radicals.
  • the radical species can be determined by the shape of the spectrum.
  • the method of the present invention it is possible to automatically determine the possibility of organopathy from the value obtained by measuring the concentration of radicals. According to the method of the present invention, it is possible to predict organopathy. More particularly, it is considered that, when organopathy is caused by factors in the blood, such factors appear prior to the actual disorder. Normal serum or plasma does not contain such factors, and thus no free radicals will be produced even if such a serum or plasma is brought into contact with the cells of the cultured organ cell lines. Only when the serum or plasma contains organopathy-inducing factors, free radicals are produced from the cultured cells. Accordingly, it is possible to automatically detect the possibility of a subject suffering from organopathy, that is, the existence of organopathy-inducing factors in the blood. Using the method of the present invention, it is possible to screen a high risk group with a high probability of disorders from a group of patients having the risk of organopathy.
  • the method for detecting of organopathy-inducing factors according to the present invention can be carried out with a small amount of a serum or plasma sample. Therefore, the present method can be used for infants or for experiments of small animals in which available sample quantity is limited. Further, because the results can be obtained in a short time, the present method is useful even for acute diseases such as acute renal failure where the state of the disease rapidly changes.
  • the measuring procedure is simple and can be carried out in a short time, and thus it is possible to treat a large amount of samples.
  • organ failure can be diagnosed in patients whose organopathy has been anticipated, before it becomes clear.
  • Transplantation of an organ such as heart, liver, or kidney, is internationally established as the treatment of organ failure, and will be accepted in Japan.
  • the most important complication in organ transplantation is an allograft rejection.
  • Acute rejection in particular is a complication often caused shortly after transplantation. The onset is rapid and, further, there is less chance of a complete recovery if treatment is started when organ failure is advanced. Therefore, early diagnosis is necessary.
  • the rise of the serum creatinine value occurs only after the decline in the kidney function proceeds to a certain extent. Also, the value fluctuates even when the kidney is normal and it is sometimes difficult to distinguish if a slight rise in the creatinine value is a normal fluctuation or reflections of the early stage of rejection. Therefore, diagnosis by some other method has been desired.
  • the present invention provides a means of solution of this problem.
  • the lymphocytes in blood are activated and the bioactive substances known as cytokines are released.
  • the bioactive substances promote the release of free radicals from the endothiel cells. Therefore, for example, using the free radical release reaction of cultured glomerular endothelial cells, caused by a serum or plasma sample, according to the present invention, it is possible to diagnose an early acute rejection reaction.
  • the method for detecting organopathy-inducing factors according to the present invention have the above characteristics, and is useful as a novel method for screening organopathy, preferably nephropathy, using EPR, and, further, is useful as a novel method for screening potential organopathy, preferably, potential nephropathy at the early stage.
  • the screening method of the present invention may be applied to a group examination to find nephropathy at the early stage.
  • the method for detecting organopathy-inducing factors according to the present invention is carried out for cells from various kind of organs as the cultured cells, it is possible to determine the organ where the organopathy occurs.
  • Example 1 EPR Patterns in Immortalized Bovine Glomerular Endothelial Cells Treated with Nephropathy-Inducing Factor
  • E. coli toxin lipopolysaccharide (LPS) (E. coli. 0111; Sigma) was used.
  • Immortalized bovine glomerular endothelial cells were prepared by the method described in Nitta K., et al., Jpn. J. Nephrol., 1994; 36: 883-889.
  • Bovine glomerular endothelial cells were separated from mature bovine kidneys, cloned, and proliferated by the modified method disclosed in Nitta K., et al., Acta Pathol., Japonica, 1993; 43: 367-371 in accordance with the method disclosed in Nitta K., et al., J. Am. Soc. Nephrol., 1991; 2: 156-163.
  • the separated GEN was maintained in a medium A.
  • the medium A was prepared by adding to RPMI 1640 medium (Gibco Oriental Co., Tokyo, Japan) 15% heat-inactivated fetal calf serum (FCS), 5 U/ml heparin, 2 ng/ml acidic fibroblast growth factor (FGF; R&D System; Minneapolis, USA), and antibiotics.
  • FCS heat-inactivated fetal calf serum
  • FGF acidic fibroblast growth factor
  • Plasmid DNA for expressing the SV40 large T-antigen (Brash DE et al., Mol. Cell Biol., 1987; 7: 2031-2034) was introduced into E. coli and proliferated. The plasmid DNA was separated by a Qiagen column. The eluted DNA was precipitated with ethanol and resuspended in sterile TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Then, the absorbance at 260 nm was measured to determine the amount of DNA.
  • the DNA was introduced into the bovine GEN by the liposome mediated method which is a modified method of the protocol of Ponder KP, et al., Human Gene Therapy, 1991; 2: 41-52 and Gibco BRL (Gaithersburg, USA).
  • the GEN was placed on a gelatin-coated six-well plastic culture plate in an amount of 2 x 104 cells/well. A cellular monolayer was formed in the medium A after 12 hours.
  • the liposome/DNA mixture was prepared by mixing 15 ⁇ g of lipofectin (Gibco BRL) and a predetermined amount of the DNA prepared above in 50 ⁇ l of distilled water. The resulting mixture was incubated at room temperature for 15 minutes, and then, added to the cultured GEN. The all preparations were incubated in 5% CO2 at 37°C for 18 to 20 hours. Then, the culture medium was replaced with a fresh medium A and the GEN was cultured for further 48 hours.
  • lipofectin Gibco BRL
  • the culture medium was replaced with a selective medium.
  • the select medium was prepared by adding to RPMI 1640 medium 10% FCS, ITS Premix (5 ⁇ g/ml insulin, 5 ⁇ g/ml transferrin, and 5 ng/ml selenium), and antibiotics. Cells which actively proliferated were selected using the usual cloning method. One clone was selected as the clone for proliferation and transferred to a 75 cm culture flask (Corning Glass Works; Corning, USA). The clone was cultured in an RPMI 1640 medium containing 10% FCS and antibiotics (medium B) and subcultured with 0.125% trypsin.
  • the culture medium used was RPMI 1640 (for example, the medium made by Gibco, ICN, or Sigma) which contained fetal bovine serum (15%), penicillin (100 ⁇ g/ml), streptomycin (100 ⁇ g/ml), and Fungizone (0.25 ⁇ g/ml) (see Yoshioka et al., Kidney International 35: 211-219, 1994).
  • the immortalized bovine glomerular endothelial cells cultured in the 6-well plate were first washed with D-PBS (D-PBS containing calcium and magnesium; Gibco). Then, a solution of lipopolysaccharide (LPS; 10 ⁇ g/ml), which usually appears in blood at the time of infection by E. coli and causes an acute renal failure, dissolved in D-PBS (0.6 ml) was added and the solution was incubated for 1 hour in a CO2 incubator (cell culture incubator: Hirasawa Seisakusho WJ-3C; Tokyo). Then, the spin-trapping agent, DMPO (Labotec, Tokyo; 0.45M, 30 ⁇ l) was added and a reaction was performed for 20 minutes. As a control test, D-PBS not containing lipopolysaccharide was added to the cultured cells. After DMPO was similarly added, the reaction was performed for 20 minutes.
  • D-PBS D-PBS containing calcium and magnesium; Gibco
  • LPS
  • the culture medium was taken in 13 x 75 mm glass test tubes (Maruem, Osaka). Then, the EPR pattern (differentiated form) of the sample was immediately determined by an EPR apparatus (JES-RE1X, Nippon Denshi). The measurement conditions were a gain of 5 x 100, a time constant of 0.1 second, a sweep of 5 mT, and a magnetic field modulation of 0.63 x 10 ⁇ 1 mT. Further, as the spin measurement standard for determining the concentration of free radicals, a standard solution (10 ⁇ 6M) of 4-hydroxy-TEMPO (made by Sigma) was prepared. The EPR pattern was measured under the conditions same as those for the sample.
  • Fig. 1 The two types of EPR patterns obtained from the above experiments are shown in Fig. 1.
  • the top pattern (control) of Fig. 1 shows the results of the control experiment, while the bottom pattern (LPS) shows the results of the experiment in which E. coli-derived lipopolysaccharide (LPS) was added.
  • the two spectra at both ends in each pattern are standard waves incorporated in the machine (Mn+ spectrum).
  • the height of the standard waves and the height of the waves obtained from the specimen indicate the relative radical concentration.
  • the concentration was calculated from the standard line of 4-hydroxy-TEMPO.
  • Fig. 1 the four waveforms observed in the pattern in the case of addition of E. coli-derived lipopolysaccharide (LPS) were spectra characteristic of hydroxyl radicals.
  • hydroxyl radicals were detected in the culture medium of the immortalized bovine glomerular endothelial cells to which one of the causative substances of acute renal failure, LPS, was added. Only minor waves were observed in the control pattern.
  • the concentrations of free radicals in the control test and the LPS-added test were 0.21 and 4.6 nmol/107 cells, respectively.
  • Example 2 EPR Patterns in Immortalized Bovine Glomerular endothelial Cells Treated with Serum of Patient Experiencing From Acute Renal Failure
  • the EPR patterns were obtained in the same way as in Example 1.
  • the release of hydroxyl radicals was observed in the immortalized bovine glomerular endothelial cells treated with the acute stage serum [top part of Fig. 2 (acute stage)], while no free radicals were detected by the serum of the convalescent stage [bottom part of Fig. 2 (convalescent stage)].
  • the amount of free radicals produced by the acute stage serum was found to be 2.4 nmol/107 cells. Further, no free radicals were detected when the acute stage serum was not contacted with the cells. This means that there exist, in the serum, factors (nephropathy inducing factors) which act on cells and promote the release of free radicals from the endothelial cells.
  • Example 3 EPR Patterns in Immortalized Bovine Glomerular Endothelial Cells and LLCPK1 Cells Treated with Serum of Patient with Acute Renal Allograft Rejection After Kidney Transplantation
  • the reactions of the initial lesions (the endothelial cells) of the rejection reaction, and the secondary lesions, (uriniferous tubular epithelial cells) of the rejection reaction, with the serum were compared using immortalized bovine glomerular endothelial cells and immortalized renal tubular epithelial cells, that is, LLCPK1 cells.
  • LLCPK1 cells Dainippon Seiyaku
  • the LLCPK1 cells as described in the specifications were subcultured in an RPMI 1640 medium containing a 10% FBS.
  • the cells used for the experiment were subcultured on a six-well culture plate in the same way as the immortalized bovine glomerular endothelial cells.
  • the serum of the early stage of the acute rejection reaction after a kidney transplantation that is, the acute stage (active) serum
  • the serum after treatment of the rejection reaction that is, the convalescent stage (inactive) serum
  • the production of free radicals was measured by the EPR.
  • Fig. 3 comparing the top part of Fig.
  • Example 4 Detection of Free Radicals Produced From Immortalized Bovine Glomerular Endothelial Cells by Serum of Acute Renal Failure Caused by Rhabdomyolysis
  • Rhabdomyolysis is a disease which causes lysis of myocytes due to the physical exertion (such as physical training or crush syndrome) in a person who has been healthy.
  • the indicator of kidney dysfunction and release of free radicals from cultured cells stimulated by the serum from the patient (12-year-old boy) were observed over time in rhabdomyolysis caused by baseball training.
  • Fig. 4 showing the results, on days after 1 and 2 days from the disease, no remarkable rise in the urea nitrogen ( ⁇ ) and creatinine ( ⁇ ) foreshadowing strong renal dysfunction necessitating dialysis was observed. Further, it is believed that the urea nitrogen and creatinine values rise along with the fusion of muscles in rhabdomyolysis. The urea nitrogen and the creatinine are contained in the protein of the muscle. Therefore, even if the kidney function does not fall, the concentration of the serum rises along with destruction of muscle. In the initial stage, these values cannot indicate the accurate kidney function. In particular, the fluctuations in the creatinine value did not suggest nephropathy. Further, while not shown in Fig. 4, the amount of urine was maintained. Thus, diagnosis of the early stage of nephropathy was difficult. Subsequently, the urea nitrogen and creatinine value rose and the acute renal failure became apparent. The patient was dialyzed on the days 4 and 5 due to severe impairment in renal function.
  • 0.1 ml of patient's serum was applied to immortalized bovine glomerular endothelial cells together with 0.5 ml of D-PBS, 30 ⁇ l of DMPO were added after one hour, and then the concentration of free radicals was measured.
  • the results are shown in Fig. 5.
  • no radicals at all were detected in the control serum ( ⁇ )
  • ⁇ in the patient's serum ( ⁇ )
  • production of over 1 nmol/107 cells of free radicals was already observed on the days after 1 and 2 days of the disease when the nephropathy was still not clear.
  • the blood was dialyzed on the days after 4 and 5 days of the disease. A fall in the urea nitrogen and the creatinine values as well as a reduction in the production of free radicals due to introduction of hemodialysis were observed.
  • the present Example shows that the method of the present invention is a useful method of diagnosis even when the organopathy is not apparent or when the nephropathy cannot be evaluated by ordinary indicators.
  • the method of the present invention is completely novel, because it can measure the organopathy-inducing factors, which were never measured by conventional methods. Thus, conventional methods can measure each factor separately, however, organ failrure often develop from multiple factors.
  • the present method can determine overall potency for sum of the factors. According to the method of the present invention, it becomes possible to judge the risk that a patient may develop organopathy, at an early stage, when the blood is taken.
  • the method of the present invention is useful for screening organopathy, preferably nephropathy using EPR, and is useful for screening potential organopathy, preferably potential nephropathy in the early stage.

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JP23432194A JPH0870889A (ja) 1994-09-02 1994-09-02 臓器障害誘導因子の検出方法
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Cited By (1)

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EP0973043A1 (en) * 1998-07-13 2000-01-19 Newpharm & Trading SA Method of diagnosing malignant neoplasms based on ESR spectroscopy

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JPH03211460A (ja) * 1989-12-31 1991-09-17 Eishun Kin フリーラジカル測定キット及び測定条件
US5091180A (en) * 1987-11-20 1992-02-25 Administrators Of The Tulane Educational Fund Protection against rhabdomyolysis-induced nephrotoxicity
WO1992018874A1 (en) * 1991-04-15 1992-10-29 The London Hospital Medical College Assessment of renal malfunction by esr spectroscopy
WO1992022290A1 (en) * 1991-06-18 1992-12-23 Oklahoma Medical Research Foundation Use of spin trapping for the treatment of diseases associated with oxidation of lipids and proteins

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Publication number Priority date Publication date Assignee Title
US5091180A (en) * 1987-11-20 1992-02-25 Administrators Of The Tulane Educational Fund Protection against rhabdomyolysis-induced nephrotoxicity
JPH03211460A (ja) * 1989-12-31 1991-09-17 Eishun Kin フリーラジカル測定キット及び測定条件
WO1992018874A1 (en) * 1991-04-15 1992-10-29 The London Hospital Medical College Assessment of renal malfunction by esr spectroscopy
WO1992022290A1 (en) * 1991-06-18 1992-12-23 Oklahoma Medical Research Foundation Use of spin trapping for the treatment of diseases associated with oxidation of lipids and proteins

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Title
DATABASE MEDLINE KNIGHT-RIDDER INFO; K. NITTA ET AL.: "Establishment and characterization of an immortalized bovine glomerular endothelial cell line" *
DATABASE WPI Week 9143, Derwent World Patents Index; AN 91-315139 *
NIPPON JINZO GAKKAI SHI, vol. 36, no. 8, TOKYO JP, pages 883 - 889 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0973043A1 (en) * 1998-07-13 2000-01-19 Newpharm & Trading SA Method of diagnosing malignant neoplasms based on ESR spectroscopy
WO2000004387A2 (en) * 1998-07-13 2000-01-27 DÜBY, Rolf, B. Method of diagnosing malignant neoplasms
WO2000004387A3 (en) * 1998-07-13 2000-10-19 Dueby Rolf B Method of diagnosing malignant neoplasms

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